HIV/AIDS is a global pandemic with 37 million individuals living with HIV infection and approximately 40 million have died from AIDS worldwide. The objectives of this project are to define the unique epidemiological, clinical, virologic, and immunologic features of HIV and its co-infections in developing countries, to determine the viral kinetics associated with sexual transmission, and to characterize the different molecular strains of HIV for infectiousness and progression of disease. We continued our research on the development, validation and application of accurate HIV cross-sectional incidence testing to determine the impact of population level interventions, identification of populations for intervention trials and epidemic surveillance. HIV-1 subtype C infects >50% of all infected individuals, and we developed an accurate multi-assay algorithm (MAA) optimized for subtype C that uses two serologic markers and viral load. This MAA had a mean window period of 248 days (95% CI: 218, 284), a shadow of 306 days (95% CI: 255, 359), and provided the most accurate and precise incidence estimate when compared to the observed incidence in HPTN 068. We determined that the Limiting Antigen Avidity Assay (LAg-Avidity) performance performed poorly on samples from Cameroon and Uganda. The subject-level false recent ratio was 5.3% (95% confidence interval CI, 2.1-10.5) for individuals infected for 1 year and 3.9% (95% CI, 0.8-11.0) for individuals infected for 2 years. These data suggest that the LAg-Avidity plus viral load incidence algorithm may overestimate HIV incidence rates in West and East Africa. We also determined that young adults infected perinatally had lower HIV-specific antibody responses as measured by HIV incidence assays than their counterparts who were infected as adults. These lower antibody responses were seen for both virally suppressed and viremic perinatally infected individuals compared with individuals who were infected as adults. This lowered marker of the anti-HIV antibody response would result in a higher frequency of long-term infected individuals being misclassified as recently infected resulting in increased estimates of HIV incidence in populations, such as South Africa, where significant proportion of these individuals occur. We investigated HCV viral clustering among and within subjects in a highly networked cohort of people who inject drugs (PWID). All subjects had estimated dates of infection and two or more E1 sequences (bp 943-1288 relative to H77) with 1 to 14years of follow up. Two methods (HIV-TRACE and PhyloPart) were used to determine clusters. Genetic distance thresholds were determined by comparing intra-and inter-host distances. Additional phylogenetic analysis was performed on subjects with complicated viral histories. At the optimal threshold of 3.9%, HIV-TRACE found 77 clusters and PhyloPart found 63 clusters, of which 27 and 32 contained multiple subjects, respectively. Furthermore, 1/3 of the subjects had sequences in different clusters over the course of the study, including some cases in which a later-sampled sequence matched a cluster detected much earlier in the infection, despite being separated by an RNA-negative lab visit and detection of sequences in different clusters. These observations suggest that subjects are either experiencing rapid and recurring infection-clearance-reinfection cycles from the same source, or a single transmission event produces a chronic infection that may go undetected and/or co-circulate with different viruses from separate transmission events. In addition to our cluster analysis we utilized longitudinal NGS data from HIV seroconverters in the Rakai cohort to examine viral evolution throughout a patients infection. These data represent the largest study to date to explore this topic and our findings indicated that HIV-1 evolves around five times faster within individuals compared to the population scale. We also demonstrated that there are two key processes driving the difference in HIV-1 evolutionary rate at the within individual and population scales. Specifically, we find support for selection against variants in the within-host viral population, which were acquired in the donor because they were beneficial in that individual, and preferential transmission of variants that are similar to those that initiated the infection. The major barrier to curing HIV infection is the persistence of HIV in latently infected resting memory CD4+ T cells. Previous work from our section and the ICER Uganda team found that the latent viral reservoir (LVR) in our Ugandan population is over 3-fold smaller than that of a previously reported American population. This represents the first quantification of latently infected resting CD4+ T cells with replication competent virus in an ART treated, virally suppressed sub-Saharan African population. We expanded on this work to design and validate a novel Bayesian model for estimating the size of the latent viral reservoir that considers the clonality of the viral outgrowth populations. This new model significantly increases the accuracy of the estimate of the infectious units per million cells (IUPM) as compared to traditional methods. It also allows for the measurement of IUPMs for individual clonal variants allowing for the longitudinal measurement of these variants overtime. We have expanded our studies examining HIV superinfection and its effects on the pandemic by exploring the role of anti-HIV neutralizing antibodies (NAb) in the context of HIV superinfection in three studies. In two collaborative projects with researchers from the MRC-Uganda and the Danish HIV cohort we explored the level of NAb in individuals prior to HIV superinfection. The initial study examined a cohort of professional sex-workers and found that HIV superinfection can occur even in the presence of detectable NAb, whereas our work in Denmark found very little NAb response prior to HIV superinfection in a small number of cases. Our third project was the first study to identify a linked heterosexual HIV superinfection event where the superinfecting partner was known. In collaboration with Dr. John Mascola and Dr. Nicole Doria-Rose from the NIAID VRC, we generated fully infectious envelope clones from this couple and examined their NAb response to each others viruses before and after HIV superinfection. In this study we found that although the female partner possessed a moderate NAb response prior to superinfection, she had no detectable response to her husbands superinfecting strain indicating that a successful HIV vaccine will need to elicit a broad NAb response to reach maximum efficacy. South Africa has the largest HIV epidemic in the world, with 19% of the global number of people living with HIV, 15% of new infections and 11% of AIDS-related deaths. In a prospective study, we screened 1714 patients in an emergency department in Eastern Cape of South Africa for HIV. Four hundred (21.6%) patients were HIV positive, including 115 (6.2%) with newly diagnosed HIV infection. The overall prevalence of HIV infection was twice as high in females (29.8%) compared to males (15.4%). These studies will be expanded in other cities identify the scope of the epidemic there and to better define the clinical cascade of care and incidence. We have also continued our work examining HIV positive to HIV positive organ transplants in South Africa and the US. As part of this later collaboration we examined the benefit of giving organs from donors that are found to be false-positive for HIV during the organ procurement process to HIV+ recipients as allowed under the HOPE act. Using data collected during our Pilot study we estimate that allowing these organs to be used will increase the overall donor pool by 50-100 donors a year.